香蕉红素氧还蛋白酵母双杂交cDNA文库的构建及鉴定

Construction and Identification of Yeast Two-Hybrid cDNA Library of Musa acuminata Rubredoxin (MaRD)

结合改良的BDSMARTTMPCRcDNASynthesisKitUserManualcDNA合成试剂盒，反转录合成带有目的接头cDNA第一链．通过LD—PCR，扩增双链cDNA（dscDNA）．产物经双酶切后回收，与经同样双酶切的酵母双杂交系统中pGADT7载体连接转化，并进行了鉴定．结果表明：提取的RAN的A200／A200=1．82，表明所提RNA纯度高；双链cDNA文库大于0．2kb，且弥散较长，成瀑布条带状；根据生长菌落计数，文库舍有1．2×10^6转化子。重组子不同插入片段在0．3—2kh之间。表明该文库达到建库要求，为下一步进行酵母双杂交试验奠定基础．此方法用于构建cDNA文库与酵母双杂交系统相结合的研究目前尚未见过相关报道．

Combination of the improved BD SMARTTM PCR cDNA Synthesis Kit User Manual cDNA synthesis kit, with the purpose of reverse transcription synthesis of cDNA first chain connector, doublestranded cDNA （ds cDNA） was amplified through LD-PCR. The product which was digested by Nde I and Cla I was connected with the pGADT7 vector which was also by the same double-digestion. The results showed that the A 200/A280 of the extracted RNA was 1.82 which means the high purity of RNA. Doublestrand cDNA library was more than 0.2 kb, growth of colony counting, 1.2×10^6 library inserts were between 0.3-2 kb, indicating and the dispersion longer falls into strips. According to the containing transformants, recombinant fragments of different that the library database meets the requirements and foundation for yeast two-hybrid test. This method for constructing cDNA library and combining yeast two-hybrid system has not been reported.